In order to achieve a functional cure for HIV infections the size of the latent viral reservoir must be sufficiently reduced to allow an indefinie cessation of antiviral drug treatment. Our strategy combines a variety of approaches that have been successfully tested against cancer and HIV-1 with the goal of achieving a significant reduction of HIV reservoirs in patients who are well suppressed on HAART. Specifically, we will combine ex vivo cytokine activation of natural killer (NK) cells with stimulation of antibody-dependent cell-mediated cytotoxicity (ADCC) using broadly neutralizing antibodies against HIV-1 Env. Env expression will be achieved by induction of proviral transcription with latency reversal drugs, such as HDAC inhibitors that are currently undergoing clinical evaluation. During the R21 phase of this application we will use newly developed ex vivo models of HIV latency to optimize the strategy. In Specific Aim 1, we will define and optimize the specificity of NK cell-mediated killing of different CD4+ T cell subsets. This will involve measuring NK cell-activating/-inhibiting receptor ligands expressed on CD4+ T cell subsets using different latency-reversing drugs, measuring the efficacy of autologous NK cells to kill latently infected CD4+ T cell subsets, and optimizing ex vivo cytokine activation of NK cells. In Specific Aim 2, we will investigate the effectiveness of using broadly neutralizing antibodies against Env to stimulate ADCC in NK cell-mediated killing of latently HIV-1-infected primary T cells, in combination with proviral reactivation. Upon completion of the R21 phase of this project we will have established reliable assays to measure NK-mediated killing of reactivated latently infected T cells, defined protocols for activating NK cells ex vivo, and identified antibodies for use in ADCC-mediated killing. The R33 phase of this application will focus exclusively on ex vivo studies using patient cells that are designed to show elimination of latently infected cells. Human subjects will be recruited from our HIV clinic, the Special Immunology Unit (SIU, UHCMC). The SIU has an active population of 1026 HIV-infected patients who are followed regularly for routine medical care; approximately 90% are receiving antiretroviral therapy (ART). In Specific Aim 3, we will validate whether techniques during the R21 phase can enhance the killing of autologous primary T cells from HIV-infected individuals. In Specific Aim 4, we will assess whether techniques developed in aims 1, 2, and 3 also lead to a reduction in the latent endogenous viral reservoir in HIV-infected patients. We will perform pairwise comparisons of NK-killing in the presence and absence of antibody for ADCC, with and without T-cell induction and with and without NK cell activation. At least 10 patient samples will be analyzed to ensure statistical significance of the results. The readout for the assays will be quantitative HIV proviral DNA and mRNA measurements from quantitative PCR reactions analyzed by next-generation sequencing. The experiments in this proposal will set the stage for clinical studies of NK-directed eradication of latent HIV. Because our strategy combines enhancing NK selectivity by ADCC using available antibodies, ex vivo activation of NK cells and in vivo activation of latent proviruses, we believe it represents a practical solution that can be readily implemented in the clinic for a large number of patients.